Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.

Masumoto, Akihide; Hemler, Martin E.

doi:10.1083/jcb.123.1.245

Cited by 109 publications

(68 citation statements)

References 83 publications

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“…Each putative cationbinding N-terminal repeat lacks a potential coordinating residue at the most C-terminal position of the cation-binding loop (26,27). One possible model is that a residue from a ligand may complete metal coordination within one of these domains to stabilize ligand binding (28), although data regarding substitution mutations in this region are inconsistent with this proposition (29).…”

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confidence: 60%

“…K562 cells were grown in RPMI 1640 (Irvine Scientific) containing 10% FCS 2 (Sigma), penicillin, and streptomycin. K562 transfectants expressing wild type ␣ 4 integrin (KA4), three derivatives harboring ␣ 4 mutations (N283E, D346E, and D408E), or vector with no insert (KpF) were as described (29). Stably transfected K562 cells were grown in media supplemented with 400 g/ml G418 (Life Technologies, Inc.).…”

Section: Bacterial Strains Plasmids and Bacterial And Eukaryotic Cellmentioning

confidence: 99%

“…␣ 4 and ␣ 3 Microtiter Binding Assay-K562 cells transfected with wild type or mutant human ␣ 4 clones were sorted with magnetic anti-mouse IgG-coated beads (Dynal Co.) coated with a 1:1000 dilution of the purified anti-␣ 4 antibody B-5G10 (ϳ1 g/ml), and expression was analyzed by FACS analysis after 2 weeks grow out as described previously (29). 96-Well microtiter plates (ICN Biochemicals) were coated with a series of dilutions of MBP-INV497 at a starting concentration of 100 g/ml (33,35).…”

Section: Bacterial Strains Plasmids and Bacterial And Eukaryotic Cellmentioning

confidence: 99%

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Differential Effects of Integrin α Chain Mutations on Invasin and Natural Ligand Interaction

Krukonis¹,

Dersch²,

Eble³

et al. 1998

Journal of Biological Chemistry

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To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human ␣ 3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the ␣ 3 chain, ␣ 3 -W220A, failed to promote adhesion to the natural ␣ 3 ␤ 1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, ␣ 3 -Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, ␣ 3 -D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-␣ 3 antibody, indicating that the second and third repeats of the ␣ 3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin ␣ chain mutations, as ␣ 4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.Invasin is a 986-amino acid outer membrane protein encoded by the Gram-negative pathogen Yersinia pseudotuberculosis (1, 2) that binds at least five different integrins containing the ␤ 1 chain, including ␣ 3 ␤ 1 and ␣ 4 ␤ 1 (3).1 Attachment to integrins by invasin leads to internalization of the bacterium (1, 2). Crossinhibition studies with human fibronectin indicate that invasin recognizes a site on the integrin identical or nearby to that utilized by natural substrates (4).Substrate recognition of natural substrates appears to involve the extracellular domains of both the ␣ and ␤ integrin chains (5-8). Each ␣ chain has a series of 7 nonidentical Nterminal repeats, approximately 60 amino acids in length ( Fig. 1; see Refs. 9 and 10). Analyses of function-blocking monoclonal antibodies and site-directed mutagenesis indicate that the third ␣ chain N-terminal repeat is required for substrate recognition (11-16). One striking feature of the identified critical ␣ chain residues found within this repeat is that aromatic side chains are important for ligand recognition (Fig. 1), reminiscent of previous observations regarding human growth hormone interaction with its receptor (17). Also found in the ␣ chain N-terminal region are three predicted divalent cationbinding motifs within repeats V-VII. Each putative cationbinding N-terminal repeat lacks a potential coordinating residue at the most C-terminal position of the cation-binding loop (26,27). One possible model is that a residue from a ligand may complete metal coordination within one of these domains to stabilize lig...

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confidence: 60%

Section: Bacterial Strains Plasmids and Bacterial And Eukaryotic Cellmentioning

confidence: 99%

Section: Bacterial Strains Plasmids and Bacterial And Eukaryotic Cellmentioning

confidence: 99%

See 1 more Smart Citation

Differential Effects of Integrin α Chain Mutations on Invasin and Natural Ligand Interaction

Krukonis¹,

Dersch²,

Eble³

et al. 1998

Journal of Biological Chemistry

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“…3 Recent studies have revealed that the ligand binding activity of integrins can be regulated by factors acting either from outside the cell or from the cell interior. These regulatory factors include temperature, 15 extracellular divalent cations, 21 integrin cytoplasmic domains, 33 and interaction with intracellular signal transduction molecules such as calreticulin, 34 serine/threonine kinases and phosphatases, 35 and members of the small GTPase family including R-Ras 36 and Rho A. 37 Especially, inside-out signaling has a critical role in integrin activation by propagating conformational changes from the cytoplasmic domains of integrins to the extracellular binding sites in response to intracellular signaling events.…”

Section: Discussionmentioning

confidence: 99%

“…The recent discovery of certain anti-␤1 antibodies, which can dramatically stimulate various ␤1 integrin functions, has made it possible to analyze the functional regulation of ␤1 integrins. 15,17,21 In the present study, we have utilized the stimulatory anti-␤1 monoclonal antibody (mAb) TS2/16 to evaluate constitutive activity of ␤1 integrins on multiple HCC cell lines. As a result, ␤1 integrins showed a wide range of constitutive activities in different HCC cell types.…”

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confidence: 99%

Role of ?1 integrins in adhesion and invasion of hepatocellular carcinoma cells

1999

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To investigate the role of integrins in hepatocellular carcinoma (HCC) invasion, we analyzed the relationship between the expression and activity of ␤1 integrins and the invasive ability of multiple HCC cell lines. Human HCC cell lines, PLC/PRF/5, Hep3B, HepG2, HLE, HuH7, and C3A cells, had high expression of ␤1 and ␣6 subunits, and various levels of ␣1, ␣2, ␣3, and ␣5 expression as determined by cell surface flow cytometry. Activity of ␤1 integrins was evaluated by cell adhesion to collagen, fibronectin, and laminin in the presence or absence of the stimulatory anti-␤1 monoclonal antibody ( Tumor invasion and metastasis are complex processes requiring the ability of tumor cells to interact with endothelial cells and extracellular matrix. The major cell surface receptors that mediate these interactions are integrins. They are heterodimeric transmembrane receptors consisting of ␣ and ␤ subunits, which produce more than 20 different heterodimers and bind specific ligands. 1,2 Each subunit is a glycoprotein with a large extracellular domain and a relatively small cytoplasmic domain. It has been shown that integrins can transduce signals and play principal roles in biological features of tumors, especially in growth, differentiation, and invasive and metastatic events of malignant cells. 2,3 Changes in expression of integrins have been linked to both tumor suppression and tumor progression in different human malignancies. 4,5 Several studies have described altered expression of ␤1 integrins in hepatocellular carcinoma (HCC). Reduced expression of ␣2, ␣3, and ␣5 subunits 6,7 and overexpression of ␣6 subunits of ␤1 integrins 8 have been shown in HCC with aggressive phenotypes. Nonpolarized aberrant distribution of ␤1 subunit has been shown to correlate with tumor growth rate, grade, and size in HCC. 9 These observations suggest important roles of ␤1 integrins in growth and invasiveness of HCC. Recently, several studies have indicated the modulation of expression and function of ␤1 integrins on HCC cells during metastatic events. Following the adhesion to vascular endothelial cells, HCC cells receive stimulation from cytokines produced by the endothelial cells. The resultant increase of ␣2␤1 integrin expression on HCC cells may facilitate migration of these cells to extravascular tissues. 10 On the contrary, the cell adhesion regulator, the gene of which is located on 16q, suppresses the process of integrin-mediated cell adhesion and has an inhibitory effect on the progression of HCC. 11

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Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Kern

Marcantonio

1998

J. Cell. Physiol.

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Adhesion to collagens by most cell types is mediated by the integrins alpha1beta1 and alpha2beta1. Both integrin alpha subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified alpha1beta1 and alpha2beta1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the alpha1 and alpha2 I domains in specific collagen adhesion. We find that introducing the alpha2 I domain into alpha1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (alpha1-2-1beta1) is similar to the adhesion profile conferred by alpha2beta1, not alpha1beta1. The presence of alpha2 or alpha1-2-1 results in preferential binding to collagen I, whereas alpha1 expressing cells bind better to collagen IV. In addition, alpha1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas alpha2 or alpha1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by alpha2beta1 or alpha1-2-1beta1, but not by alpha1, requires the presence of Mn2+ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins.

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Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.

Cited by 109 publications

References 83 publications

Differential Effects of Integrin α Chain Mutations on Invasin and Natural Ligand Interaction

Differential Effects of Integrin α Chain Mutations on Invasin and Natural Ligand Interaction

Role of ?1 integrins in adhesion and invasion of hepatocellular carcinoma cells

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

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